Anthrax toxin protective antigen (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then binds to the other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis via lipid rafts and pass through endocytic vesicle populations, and then translocate LF and EF to the cytosol. EF is a calcium and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Anthrax lethal toxin (LT) plays a central role in the virulence of Bacillus anthracis and causes profound effects on cultured cells and in experimental animals. Our laboratory previously reported extensively on the vascular collapse induced by LT in mice, although the organ or cellular target for the toxin was unknown. In the current period, new electron microscopic and cardiac biomarker studies have identified the heart as the primary target of this toxin. LT targets murine myocardial cells and the cardiac endothelium within hours of administration to mice. Furthermore, inbred knockout mouse models allowed us to show a protective role for neuronal nitric oxide synthase (nNOS) against toxin-mediated effects in the mouse. Collaborator laboratories have also verified the targeting of the heart in the rat toxin challenge model. Norepinephrine therapy, normally helpful in most bacterial shock, was shown to be ineffective against LT in the rat model, supporting the hypothesis that LT causes direct cellular injury insensitive to vasopressor effects. Our findings, coupled with those of two collaborating laboratories have now confirmed the unique shock induced by LT as likely to be a downstream effect of toxin targeting of the heart. A striking effect of LT in cells is the rapid lysis this toxin induces in murine macrophages. This lysis requires activation of caspase-1 by the Nlrp1 inflammasome. Our previous studies detailed the kinetics of signaling events leading to lysis, and showed a role for the proteasome in LT-mediated inflammasome activation. In the current period evidence was obtained showing that LT activation of the NLRP1b inflammasome involves lysosomal membrane permeabilization and cytoplasmic cathepsin B activity. The cathepsin B inhibitor CA-074Me functions downstream of LT-mediated MEK cleavage, K(+) effluxes, and proteasome activity in preventing caspase-1 activation by the toxin. These findings help to characterize the mechanistic pathways by which LT induces cell death. Finally, we have worked with collaborators at the FDA to develop better diagnostic tools for detection of anthrax infections. A highly specific europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax toxin components (100-fold more sensitive than standard ELISA methods used to detect toxin) was developed and verified in a real infection model. The development of these and future ENIA diagnostic tools should facilitate the sensitive detection of B. anthracis bacteria and the diagnosis of anthrax infections at earlier stages when antibiotic and supportive therapy may still be effective.